This project is aimed at the development of mass spectrometry-based assays for the identification of small- molecule ligands capable of disrupting the binding of HIV-1 nucleocapsid (NC) protein with stemloop domains of the genome packaging signal ((-RNA). These specific protein-RNA and RNA-RNA interactions mediate critical functions in the process of genome recognition, dimerization, and packaging, which constitute ideal targets for the development of new therapeutic strategies. With the goal of identifying small-molecule candidates that may disrupt such processes, we propose to employ electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry to determine the effects of potential ligands on these specific interactions. The proposed assays will require simple mixing of prospective ligand with preformed protein-RNA and RNA-RNA assemblies. After binding equilibria are re-established, the unique molecular masses and isotopic compositions characteristic of the different complexes in solution will allow for their unambiguous identification by direct ESI-FTICR analysis, thus providing an immediate reading of the assay output. The selected analytical platform will enable the exploration of the widest possible chemical space with no need for the presence of chromophores or radioactive labels. The absence of derivatization and separation procedures will not only expedite analysis, but will also minimize the risk of artifactual results. The pilot study will define the conditions for completing the assays and will evaluate their potential for high-throughput primary screening of extensive ligand libraries. It is expected that this protocol will provide a rapid and sensitive approach for examining compounds included in the NIH Molecular Libraries Small Molecule Repository (MLSMR) to identify possible leads for cell-based secondary screening. 1 The growing threat of drug resistant strains has greatly increased the urgency of the search for new therapeutic strategies to control the HIV pandemic. Due to their critical functions in the virus life cycle, the assemblies formed by the nucleocapsid (NC) protein with stemloop domains of the genome packaging signal ((-RNA) constitute ideal targets for intervention, which are still untapped. The highly specific nature of the interactions between NC and the different stemloops offers the opportunity of inhibiting such functions without affecting normal host processes. The proposed assays will provide a robust tool for exploring the chemical space in search of small-molecule ligands capable of disrupting these interactions. The flexibility afforded by the selected analytical platform will enable the detection of species that may bind to target assemblies without inducing dissociation, thus expanding the scope of the information attainable from the assays. Applied to large compound libraries, these assays are expected to enable the identification of small molecules that may serve as leads for the development of inhibitors and diagnostic tools targeting the processes of genome recognition, dimerization, and packaging in HIV-1. 1 [unreadable] [unreadable] [unreadable]